1. Field of the Invention
The present invention relates to an organic polymer separation membrane having a fluorene skeleton. More specifically, it relates to a separation membrane having an excellent heat resistance and mechanical strength, and an oxygen enrichment device for cell culturing utilizing the membrane.
2. Description of the Related Art
Recently, separation techniques utilizing various organic polymer membranes such as a microfiltration membrane, ultrafiltration membrane, reverse osmosis membrane, and gas selective permeation membrane have been remarkably developed and some of these membranes are now practically used as an industrial scale. For example, microfiltration membranes or ultrafiltration membranes are used for the treatment of industrial waste liquids, the recovery of the waste water from buildings, and the concentration of liquid foods and reverse osmosis membranes are used for desalination of sea water.
However, the above-mentioned separation membranes have a poor heat resistance, and therefore, separation membranes for high temperature fluid is still under research and separation membranes capable of affecting a high pressure steam sterilization treatment requiring higher heat resistance have not been proposed. Regarding gas selective permeation membranes, i.e., membranes for enriching a specific gas in a gas mixture, although membrane permeation of gases having largely different molecular weights such as, for example, hydrogen gas and methane gas are being practically used, the practical use of oxygen enrichment membranes capable of obtaining air having an enriched oxygen concentration in the medical field is limited.
There are many industrial utilization fields of oxygen enriched air in which a high oxygen concentration is not necessarily required. For example, an oxygen enriched air containing 25% to 30% oxygen can be used for, for example, combustion furnaces such as blast furnaces and high temperature gas furnaces. However, in these application fields, the oxygen enriched air must be stably supplied in a large amount and at a low cost.
The technical problems to be overcome when an oxygen enriched air is obtained by a gas selective permeation membrane is how to increase the permeation flow rate per unit area of the membrane without decreasing the selective permeability. Furthermore, when a gas selective permeation membrane is practically arranged as a module, the membrane area per unit volume of module should be increased.
In addition, since there are inverse relationship between the thickness of a membrane and the permeation flow rate per unit area of the membrane, the thickness of the gas permeation membrane should be decreased. As a preparation method of such an oxygen enrichment membrane, it has been proposed to produce a composite of a thin film or membrane having a permeation or separation function and a supporting porous membrane. For example, it has been proposed in Japanese Unexamined Patent Publication (Kokai) No. 54-40868 that a gas permeation membrane having a very thin thickness (e.g., about 0.1 .mu.m) is formed by dropwise applying, for example, an organopolysiloxane.polycarbonate copolymer solution on the surface of a liquid casting support, whereby a composite with a porous support is formed.
However, it is very difficult to prepare the above-mentioned composite membrane having a very thin separation or permeation membrane on the surface thereof, without causing any defects such as cracks and pin holes. Furthermore, the handling during the preparation of the module is trablesome and other problems also arise. Moreover, the above-mentioned siloxane type separation or permeation membranes have a disadvantageously poor heat resistance and the optimum gas permeation temperature is about 20.degree. C. and the gas permeation or separation ratio is decreased with the increase in the temperature. For example, at a temperature of 40.degree. C. or more, the membrane does not desirably function as an oxygen enrichment membrane.
The conventional production steps of wet process methods of preparing hollow fiber membranes generally comprise a membrane fabrication dope preparation step for dissolving the membrane base polymer in a solvent; a coagulation core liquid preparation step, an extruding step for extruding the membrane fabrication dope and the coagulation core liquid through an annular orifice and a central circular orifice of a double tubular spinning nozzle, respectively, a an air gap for sending the extruded products from the spinning nozzle to a coagulation bath through a surrounding air atmosphere (i.e., an air gap), and a coagulation step for effecting the solvent removal and coagulation. The crosssectional structure of the separation or permeation membranes obtained by the above-mentioned production steps usually comprise a so-called surface dense layer (i.e., micropore layer) for determining cut-off molecular weight and a porous layer for supporting the micropores.
Attempts have been made to prepare hollow fiber separation membranes having a pore size of 0.01 .mu.m or more and suitable for use as a ultrafiltration membrane and microfiltration membrane by appropriate production steps. However, there are still requirements for improving the adjustment or control of the size of the surface micropores and the mechanical strength of the hollow fibers. For example, as is disclosed in Japanese Unexamined Patent Publication (Kokai) No. 55-106243, polyethylene oxide is added to the membrane fabrication dope, after the membrane is fabricated, but it is still difficult to obtain the desired hollow fiber separation membrane having a uniform pore size of 0.01 .mu.m or more. Furthermore, a hollow fiber membrane having a pore size of 0.01 .mu.m is prepared from polysulfon as a base material by the phase separation phenomenon of the membrane fabrication dope, as disclosed in Japanese Unexamined Patent Publication (Kokai) No. 60-222112. However, the viscosity of the membrane fabrication dope is low and it is difficult to prepare hollow fiber separation membranes having a satisfactory membrane thickness. In addition, these hollow fiber separation membranes have no heat resistance sufficient to withstand against high pressure steam sterilization conditions.
On the other hand, in the growth and proliferation of animal cells, plant cells, and aerobic microorganisms in a cell culture broth in the form of a liquid or suspension, it is necessary to feed nutrient components, remove harmful metabolites, and feed oxygen, together with the provision of a solid surface corresponding to the demand of the cells, and these constitute the cell culture limiting factors. Accordingly, to realize an industrial bulk and high density culture of cells and microorganisms, the obstacles represented by these culture limiting factors must be removed. Further, among these limiting factors, an improvement of the feeding of oxygen is most strongly required.
The oxygen concentration in the culture broth, namely the dissolved oxygen concentration, is at a maximum at the equilibrium solubility of the oxygen, which is determined by the oxygen partial pressure in the gas phase, when a temperature and a culture broth composition are given. Nevertheless, since the cells in a culture broth constantly consume oxygen, the dissolved oxygen concentration is smaller than the equilibrium solubility, and it becomes necessary to ensure a stable feed of oxygen, for the growth and proliferation of cells.
On the other hand, the cell culturing method is intended for the growth and proliferation of only a specific cell species, and any mixing of heterologous cells of miscellaneous microorganisms must be avoided. In other words, prior to the introduction of the desired cell species, the culturing device and the culture broth must be brought to a completely sterile and organism-free state. As such a sterilization method, there are known sterilization with ethylene oxide, chemical sterilization with an aqueous hyprochlorite solution, or high pressure steam sterilization. Of these sterilization methods, the high pressure steam sterilization method is industrially most preferable because washing or waste disposal after the sterilization operation is not required. Accordingly, the method of feeding oxygen into a cell culture must be based on a method which enables one of these sterilization methods, preferably the high pressure steam sterilization operation, to be used.
As the method of feeding oxygen into a culture broth, usually a bubble aeration of air from which microorganisms have been removed by a filter is employed. In this bubble aeration of air, since a dissolution of oxygen occurs at the bubble surface when rising through the liquid, fine bubbles alone must be generated in accordance with an increase of the aeration amount. Also, although it is possible to enhance the dissolved oxygen concentration by a bubble aeration of separately prepared oxygen-enriched air with a higher oxygen concentration than that of normal air, most of the enriched air will be released without dissolution, and thus this method is not industrially practical.
In cell cultivation, bubble aeration is the simplest way of feeding oxygen, wherein high pressure steam sterilization is feasible, but some problems to be solved remain, as shown hereafter. First, an excessive foaming accompanies bubble aeration. Generally, a cell culture broth is a highly viscous liquid, and bubbles introduced from the bottom of a culture vessel exist on the surface of the culture broth for a long time before collapsing, and consequently, a larger culture vessel volume is required compared with the culture broth volume. Second, cells coming into contact with the bubbles during the process of the generation of bubbles at the bottom of the culture vessel and the rise thereof through the liquid until finally collapsed, are damaged. Particularly, in animal cells in which the cell membrane has a poor mechanical strength, serious damage by contact with bubbles has been observed. Third, the bubble aeration is accompanied by a loss of the culture broth due to evaporation. This loss is considerable in the cultivation of animal and plant cells over a long term cultivation period and it becomes necessary to appropriately supplement water and the culture broth to correct the concentration of nonvolatile components contained in the culture broth and counteract a lowering of the liquid level.
As the means for solving the problems accompanying bubble aeration as mentioned above, various methods have been proposed in the prior art. For example, in Japanese Unexamined Patent Publication (Kokai) No. 62-195276, a method of carrying out cultivation while blowing a gas containing oxygen onto the culture broth surface is proposed. Such blowing gas feeding method, although having the advantage of eliminating the problems accompanying the generation of bubbles, provides a limited contact surface between the gas and the culture broth at the culture broth surface, whereby a deficiency in the amount of the gas supplied by an increase in the depth of the culture vessel occurs, and therefore, it is difficult to increase the scale of the cell cultivation.
The method of feeding oxygen over a large contact area between the gas and the culture broth, in which the generation of bubbles is inhibited, comprises diffusing a gas containing oxygen into a culture broth through a membrane dipped in the culture broth. For example, Japanese Unexamined Patent Publication (Kokai) No. 61-100190 has proposed a bubbleless gas feed method and device for feeding gas into the culture broth through a porous hollow fiber membrane not having a dense surface layer. Nevertheless, since such a porous hollow fiber membrane does not have an oxygen enrichment function, separately prepared oxygen-enriched air must be supplied to increase the amount of dissolved oxygen.
As the method of feeding gas into a culture broth by using a material having an oxygen enrichment function, Japanese Patent Publication (Kokoku) No. 60-23834 discloses a culturing method in which gas is diffused into the culture broth through a tube made of silicone rubber. But, since a thick tube is used, a high air permeation rate cannot be obtained, and thus, the gas to be fed is limited to oxygen. Also, it is difficult to increase the scale of the cell cultivation.
On the other hand, as an oxygen enrichment membrane having an excellent oxygen enrichment performance and a high gas permeation rate, Japanese Patent Publication (Kokoku) No. 59-51321 proposed a composite membrane of a polysiloxane type thin film and a porous carrier, which has been practically applied as an oxygen enrichment instrument. Although it is possible in principle to dip such a polysiloxane type oxygen enrichment film in a cell culture broth, thereby converting normal air to oxygen-enriched air and feeding gas under bubbleless conditions, due to the low heat resistance of the polysiloxane thin film, the device and the culture broth cannot be sterilized by pressurized steam in an autoclave, and therefore, it is difficult to widely utilize this method for feeding oxygen-enriched air in cell cultivation.
As apparent from the above description of the oxygen feeding means of the prior art in the cell culturing method, a gas feeding method which can enhance the dissolved oxygen concentration in the culture broth and at the same time avoid problems caused by bubble generation, and further, is industrially feasible and can be sterilized by high pressure steam, has not been proposed.